Process of splitting steroids



3,070,513 PROCESS OF SPLITTING STEROIDS Harry A. Toulmin, Jr., Dayton, Ohio, assignor to Basic Research Corporation, Dayton, Ohio No Drawing. Filed May 17, 1960, Ser. No. 29,579 Claims. (Cl. 195-51) This invention relates to enzymatic processes for the splitting and destroying of steroids, sterols, lipins and the like complex polycyclic compounds containing 25 or more carbon atoms in the molecule. The process of the invention is exemplified in the splitting and destruction of sterols such as cholesterol and solid fatty alcohol derivatives of cyclopententenes, phenanthrene, and the like, which are relatively stable substances and diflicult to degrade or hydrolyze.

While it is known that sterols are converted to their simple elements by microorganisms present in soils, little is known of the degrading microorganisms responsible for this action or the full nature of the reaction which takes place.

It is also known that sterols such as cholesterol can be oxidized with strong dichromate-sulfuric solutions to form the corresponding lower molecular weight ketones as reported by Leobisch, Doree and Diels. (See Ber. 5, 510

(1872); Soc. 1328 (1908); C. Doree ibid, 95, 639 (1909), v

and Ber. 41, 2596 (1908).) Further, Windaus and Resau, Ber. 46, 1246 (1913), found that the oxidation of cholesteryl acetate in boiling acetic acid and chromic acid produced methyl isohexyl ketone. Such processes have the disadvantage, however, that corrosive acids are required and it is difficult to control the action to produce the desired lower molecular weight'compounds.

Enzymatic splitting processes for producing fatty acids and glycerol from fats, e.g., fatty acid triglycerides in the manufacture of soaps are also well known. Enzymes also have been found which are useful for splitting acyl amino acids. For example, benzoyl amino propionic acid has been reported to be split by the action of hippuricase enzyme to give benzoic acid (Neuberg and K. Linhardt, Biochemical Zeitschr. 147: 372476 (1924). Fatty acid glycerides and amino compounds, however, are relatively easily degraded, but such processes cannot be used successfully to split :a sterol such as cholesterol which has been found to remain unchanged after boiling in aqueous potassium hydroxide solution for hours.

The prior art processes thus fail to provide a method of splitting and destroying these poly-ring steroid-type compounds by enzymatic catalysis. Moreover, prior processes are applicable only to in vitro-methods whereas the enzymatic process of this invention may be employed for splitting and destroying sterols in vitro or in vivo treatment methods. More recently, it has been proposed to utilize organic solvents which dissolve these sterols, e.g., choles terol, but such compounds are more or less toxic and furthermore do not eliminate the sterols but merely put them in solution.

It is the principal object of the present invention to provide a process for splitting and completely destroying steroids, e.g., cholesteryl acetate, and sterols, especially cholesterol, ergosterol, lanosterol, stigmasterol and the like poly-ring compounds of the monohydroxy secondary alcohol type which are difficult to split into lower acyclic chain compounds.

It is a further object of the invention to provide an improved process of splitting these complex compounds by treatment of the same to a powerful enzymatic action which splits open the ring structure and breaks up the compound into lower molecular weight acyclic components.

Cholesterol is probably best known of the sterols. This 3,970,513 Patented Dec. 25, 1962 is because it has been found to be the predominant constituent of human gall stones deposited in the bile duct. It has the empirical formula C H OH and in crystalline form melts at C.

The structural molecular configurations of cholesterol as well as ergosterol, lanosterol and stigmasterol are shown in the formulae:

H C CH CH CH CH CH Tl- CH 3 CHOLESTEROL CH Hgc 3 ERGOSTEROL LANOSTEROL Stigmasterol e a; In accordance with the present invention, a new enzy- EXAMPLE A steroid ENZYM ATIC CATALY$lS degradation pro due to ACYCLIC COMPOUNDS EXAMPLE B sterols \(VY Enzymatic catalysis acyoli 0 compounds In view of the high specificity of enzymes, it is essential to employ the proper enzyme or combination of enzymes to carry out the treatment under conditions which produce the powerful splitting action required to degrade the complex cyclo organic compounds. To accomplish this two types of enzymes are employed, one which breaks or splits the cycle-ring structure and another which attacks and splits the hydrocarbon chain structure. Preferably the enzymatic action or degradation is carried out concurrently using a suitable combination of the enzymes to obtain a synergistic enzymatic action. The reaction, however, may, be carried out as coordinated steps whereby the sterol compound is subjected to the concentrated enzymatic action to break the cycle-ring structure followed by further enzymatic treatment to split the hydrocarbon chain structure resulting in the production of degraded, lower molecular weight compounds and ultimately openchain or acyclic organic compounds.

A powerful enzyme capable of attacking the cyclostructure of sterols and splitting the same is an enzyme derived from Aspergillus wentii.

The preparation of a suitable enzyme from the mold Aspergillus wenlii is described in the US. patent of Carlson et al., No. 2,709,150. The method as therein disclosed comprises growth of the mold and production of the enzyme in the presence of a nutrient medium whose active ingredients may comprise a combination of peptone and a nutrient salt medium.

A typical nutrient medium which causes the mold to grow rapidly within a few days to produce a solution rich in dextranase enzymes is as follows.

Distilled water, 1 liter.

Additional examples include: (1) a medium of the composition given above, with B-vitamins omitted; (2) a medium with the composition given above, but with the B-vitamins replaced by yeast extract, 0.4 gm. per liter; (3) a medium of the composition given above, but with the peptone replaced by casein hydrolysate 5.0 gm. per liter, either with or without the B-vitamins; (4) a medium of the composition given in which the dextran component is varied from 5.0 to 50.0 gm. per liter. In preparation of the enzyme-containing medium the inorganic salts are dissolved in the Water and sterilized by heating and then after adding the other components the pH of the medium is adjusted to between 7.2 and 7.6 by the addition of a sterile solution l-N sodium hydroxide solution. Use of peptone or casein hydrolyzate together with dextran salt solutions results in enhanced stimulation of the endo-dextranase from the Aspergillus wentii mold.

Enzyme B To provide an enzyme which vigorously attacks the Components: Grams/ liter KH PO 0.5 (NHQ SQ; 0.5 MgSO.; 0.2 CaCl 0.01 FeSO .7H O 0.005 MnSO 0.002 Na MoO .2H O 0.002 Na CO 0.1

Distilled water, 1 liter.

In preparation of the medium, the inorganic salts are dissolved in the water and the solution sterilized by treating and then the medium is adjusted to a pH of between 7.2 and 7.6 by the addition of l-N sodium hydroxide solution.

In carrying out the splitting action of the invention concentrations of the enzymes of 5 to by weight of the mass or solution are preferably employed to treat the sterols. The invention will be more clearly understood from the following examples which exemplify the splitting action of the enzymes.

EXAMPLE I To a 10% sterile solution of cholesterol dissolved in benzene is added a portion of the medium obtained from the growth of Aspergilius wentii prepared as described under Enzyme A. Approximately 10 cc. of the clear filtrate is added to 100 cc. of the cholesterol solution and incubated for six hours at 27 C. Thereafter the reaction solution is filtered and to the filtrate containing partially degraded cholesterol there is introduced 5% by volume of the culture medium containing Agrobacterium etlumicm, and prepared as described in Example B. The enzymatic catalysis is continued at 30 C. for another six hours to complete the degradation of cholesterol to acylic compounds including beta-hydroxy-beta-methyl-glutaric acid.

EXAMPLE II The enzymatic catalysis is repeated as set forth in Example I, but using ergosterol to produce acyclic degraded products thereof.

EXAMPLE III The enzymatic catalysis in accordance with Example I is repeated using squalene to produce levulinic acid, succinic acid and acetone.

EXAMPLE IV EXAMPLE V In this instance lanosterol is substituted for cholesterol 6 and the enzymatic catalysis carried out in situ employing the combination of Aspergillus wentii and Agrobacterium ethanicus, as described in Example IV. Degradation of the lanosterol to C C and C acyclic compounds is thus efiected. v

EXAMPLE VI In this instance stigmasterol was split to produce lower molecular weight acyclic components when treated as described in Example I.

EXAMPLE VII Example IV was repeated using cholestryl acetate in place of cholesterol to produce lower molecular weight short-chain aliphatic compounds.

In place of employing the enzyme culture mediums as described, the enzyme may be added as a solid, dry stable form by precipitaton of the enzyme from the culture medium. This may be accomplished by adding sodium carboxymethyl cellulose (CMC) dissolved in ethyl alcohol (40 gms. CMC per 100 gms. alcohol). The CMC and enzyme precipitate is allowed to settle, and the supernatant liquor is drawn off and the solids which contain the enzyme is filtered out and after washing with ethyl alcohol dried in vacuum at about 30 C. to obtain a dry powder comprising the enzyme.

In the enzymatic catalysis action the degree of splitting and destruction of the steroid or sterol is determined principally by the enzymes employed and length of time they are allowed to act on the substance to be split. The pH of the enzyme-containing reaction mixture is also a factor and in general should be maintained on the alkaline side, and between 7.2 and 9.0 pH, at pH of 7.8 being preferred to speed up the catalysis action. The concentration of th enzyme is not critical so long as sufficient amount is present to bring about the splitting action. An enzyme concentration of one part in one thousand on weight of sterol treated is effective but somewhat higher concentrations are preferred to accelerate the catalysis.

Various substitutions and changes may be made in the process by those skilled in the art without departing from the spirit and scope of the invention.

What is claimed is:

1. A process of splitting and destroying steroids selected from the group consisting of cholesterol, ergosterol and lanosterol into lower molecular weight compounds which consists in dissolving the steroid in solvent and reacting the same with cultures of Aspergillus wentii and Agrobacterium ethanicus which splits the ring structure of the steroid molecule into hydrocarbons of lower molecular weight, and then continuing the catalysis in the presence of an enzyme which splits the lower hydrocarbons into acyclic hydrocarbons.

2. A process of splitting and destroying cholesterol into lower molecular weight compounds which consists in dissolving the cholesterol in solvent and reacting the same with cultures of Aspergillus wentii and Agrobacterium ethanicus which splits the ring structure of the cholesterol molecule into hydrocarbons of lower molecular weight, and then continuing the catalysis in the presence of an enzyme which splits the lower hydrocarbons into acyclic hydrocarbons.

3. A process of splitting and destroying ergosterol to produce acyclic degraded compounds therefrom which comprises dissolving ergosterol in solvent and reacting the said ergosterol with a culture of Aspergillus wentzi, and continuing the catalysis in the presence of a culture of Agrobacterium ethanicus to bring about splitting of the ergosterol into lower molecular weight acyclic com pounds.

4. A process of splitting and destroying lanosterol into lower molecular weight compounds which comprises dissolving lanosterol in hydrocarbon solvent and thereafter introducing cultures of Aspergillus wentii and Agrobacterium eI/Icmicus and heating the resultant mixture for several hours at a temperature of 28 C. to effect the splitting and destroying action and produce lower acyclic hydrocarbon compounds.

5. A process of splitting and destroying lauosterol which comprises dissolving lanosterol in hydrocarbon solvent and then first treating the same with a culture of References Cited in the file of this patent UNITED STATES PATENTS Taggart Mar. 19, 1946 Carlson et a1. May 24, 1955 

1. A PROCESS OF SPLITTING AND DESTROYING STEROIDS SELECTED FROM THE GROUP CONSISTING OF CHOLESTEROL, ERGOSTEROL AND LANOSTEROL INTO LOWER MOLECULAR WEIGHT COMPOUNDS WHICH CONSISTS IN DISSOLVING THE STEROID IN SOLVENT AND REACITNG THE SAME WITH CULTURES OF ASPERGILLUS WENTII AND AGROBACTERIUM EHTANICUS WHICH SPLITS THE RING STRUCTURE OF THE STEROID MOLECULE INTO HYDROCARBONS OF LOWER MOLECULAR WEIGHT, AND THEN CONTINUING THE CATALYSIS IN THE PRESENCE OF AN ENZYME WHICH SPLITS THE LOWER HYDROCARBONS INTO ACYCLIC HYDROCARBONS. 